EFFECTS OF IRON AND COPPER DEFICIENCY ON THE ...

J. Phycol. 46, 974–981 (2010) 2010 Phycological Society of America DOI: 10.1111/j.1529-8817.2010.00884.x

EFFECTS OF IRON AND COPPER DEFICIENCY ON THE EXPRESSION OF MEMBERS OF THE LIGHT-HARVESTING FAMILY IN THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) 1 Song-Hua Zhu Department of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4

Jian Guo, Maria T. Maldonado Department of Earth and Ocean Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1Z4

and Beverley R. Green2 Department of Botany, University of British Columbia, Vancouver, BC, Canada V6T 1Z4

Iron plays a crucial role in many core biochemical processes that require electron transfer reactions, such as photosynthesis, respiration, and nitrogen assimilation (Geider and Laroche 1994). It has been estimated that 80% of the iron required by phytoplankton is allocated to the photosynthetic apparatus, since there are at least 20–23 atoms of iron in a linear photosynthetic electron transport chain (Raven 1990, Raven et al. 1999). Copper is also vital for phytoplankton growth. It is involved in photosynthesis (i.e., copper-containing plastocyanin) in many cyanobacteria and chl b–containing algae (Sandmann et al. 1983) and in respiration (cytochrome oxidase) in all phytoplankton (Stryer 1988). This element also participates in detoxification of active oxygen species via Cu-containing superoxide dismutase and ascorbate oxidase (Chadd et al. 1996, Merchant et al. 2006). Furthermore, recent studies have shown that a copper-containing ferroxidase is involved in the high-affinity iron transport system of phytoplankton (La Fontaine et al. 2002, Peers et al. 2005, Maldonado et al. 2006). Although iron is the fourth most abundant element in the earth’s crust, dissolved Fe is present at subnanomolar levels in surface waters of the open ocean (Johnson et al. 1997). Indeed, numerous studies have demonstrated that iron plays a central role in controlling phytoplankton growth in 50% of the global ocean, especially in the ‘‘high nutrient, low chlorophyll’’ (HNLC) regions of the subarctic Pacific, equatorial Pacific, and the Southern Ocean (Martin et al. 1990, 1994, Bruland et al. 1991, Debaar et al. 1995, Coale et al. 1996, Boyd et al. 2000, Tsuda et al. 2003). It has also been demonstrated that copper limitation results in decreased rates of iron uptake in coastal and open-ocean diatoms, suggesting that there is an interactive effect between copper and iron nutrition (Peers et al. 2005). The completed genome sequence and the availability of a large number of EST sequences (Armbrust et al. 2004, Maheswari et al. 2005) have

The LI818 proteins and their Lhcx homologs in diatoms are a subgroup of the light-harvesting (LHC) antenna family, suspected of being involved in photoprotection and stress resistance. In this work, we report that the transcription of three LI818–like genes in Thalassiosira pseudonana Hasle et Heimdal (Lhcx1, Lhcx5, and Lhcx6) was down-regulated under iron or copper deprivation and when both trace metals were limiting, as was the case for Lhcf4, one of the standard light-harvesting genes. By contrast, the protein encoded by Lhcx1 was clearly up-regulated under iron limitation, suggesting that this gene is independently regulated at transcriptional and translational levels. In general, copper starvation had less effect on the expression of lightharvesting protein genes than iron deprivation, reflecting the different roles of iron and copper in photosynthetic function, that is, as an essential part of the electron transport chain versus as a cofactor for enzymes required to deal with the reactive oxygen species that result from inhibition of electron flow. Our results suggest that the Lhcx1 protein may be involved in stabilizing the photosynthetic apparatus when decreased nonphotochemical quenching (NPQ) results from Fe deficiency. Key index words: copper deficiency; diatom; gene expression; iron deficiency; Lhcx genes, LI818; photoprotection; photosystem I; Thalassiosira pseudonana Abbreviations: FCP, fucoxanthin chl a ⁄ c protein; F v ⁄ F m, maximal quantum yield of PSII; LHC, light-harvesting complex; NPQ, nonphotochemical quenching; RC, reaction center

1

Received 25 August 2009. Accepted 19 April 2010. Author for correspondence: e-mail [email protected].

2

974

975

F e - C u D E F I C I E N C Y A N D L h c E X PR E S S I O N

facilitated the identification of genes in T. pseudonana (CCMP1335). T. pseudonana has genes for 32 members of the LHC protein superfamily, belonging to three distinct clades: the major ‘‘standard’’ Lhcf proteins (also called fucoxanthin chl a ⁄ c proteins [FCPs] in other heterokont algae), the red algal–like Lhcr proteins, and the LI818-like Lhcx proteins (Green 2003, 2007). The LI818 gene was originally discovered in Chlamydomonas as an LHC homolog with a unique expression pattern (Gagne and Guertin 1992, Savard et al. 1996). LI818-like homologs have been observed in green algae and algae with chl a ⁄ c lightharvesting antennas but are not found in red algae or higher plants (Koziol et al. 2007). Remarkably, these proteins are the only members of the LHC superfamily shared between the green lineage (containing chl a ⁄ b LHCs) and the red lineage to which diatoms belong (Green 2003). The three Chlamydomonas reinhardtii LI818 genes are highly induced under various stress conditions, including high light and iron deficiency (Im et al. 2003, Zhang et al. 2004, Moseley et al. 2006, Naumann et al. 2007, Yamano et al. 2008), and two of the three LI818 proteins are strongly up-regulated under iron limitation (Naumann et al. 2007). The genome of the centric diatom T. pseudonana encodes five LI818 homologs, which have been designated as Lhcx1, Lhcx2, Lhcx4, Lhcx5, and Lhcx6. Lhcx1 and Lhcx2 are so closely related that their transcripts (and proteins) cannot be distinguished experimentally, so they are considered together as ‘‘Lhcx1.’’ Consistent with the work in C. reinhardtii, transcription of all but one of the Lhcx genes is highly induced by high-light stress (Zhu and Green 2008, 2010), and the accumulation of two of the proteins is increased. The Lhcx genes therefore appear to be stress-response members of the LHC superfamily, but little is known about the effect of iron and copper deficiency on their expression in diatoms. In the present study, we examined the expression pattern of the Lhcx genes in cultures acclimated to iron and copper deficiency, using quantitative real-time PCR (qRT-PCR), and compared it to the expression of several Lhcf genes. The abundance of the protein encoded by Lhcx1 was followed

by separating proteins on SDS-polyacrylamide gels and immunoblotting with a specific antibody. In addition, the maximum PSII efficiency and NPQ were measured to assess the effect of trace-metal deficiency on the PSII reaction center and the photoprotection capacity of T. pseudonana under excess light. MATERIALS AND METHODS

Culture conditions. An axenic culture of T. pseudonana (CCMP1335) was cultured in sterile artificial seawater medium AQUIL (Price et al. 1988 ⁄ 1989) with various additions of iron and copper (Table 1), under continuous light at 150 lmol photons Æ m)2 Æ s)1 at 19 ± 1C. Except for the addition of iron and copper, the AQUIL medium used in this study was prepared and had identical chemical composition as that described in Maldonado et al. (2006). Sterile and trace-metalclean techniques were used during all manipulations. Cultures were acclimated to various Fe and Cu levels in 28 mL polycarbonate tubes using semicontinuous batch cultures. Cultures were considered acclimated when the growth rates of five successive transfers varied by 2-fold compared to the Fe-sufficient control (Table 1). The growth rate of cells grown under the low-Fe ⁄ low-Cu condition also decreased 2.5fold. By contrast, the growth rate of cells grown under low Cu did not change, indicating that iron starvation was the cause of decreased cell division. In addition, the cell size was decreased by 17%–19% in the low-Fe and low-Fe ⁄ low-Cu treatments compared to the control, but no significant change was observed when cells were exposed to low Cu alone. The total cellular protein concentration varied among different treatments, with the low-Fe treatment exhibiting the lowest content (1.93 pg Æ cell)1). However, when normalized to cell volume, the protein concentration was approximately the same (0.070 pg Æ fL)1) for all treatments except for the low-Fe ⁄ low-Cu treatment. Similarly, total chl per unit cell volume was effectively unchanged, suggesting that there was no overall down-regulation of the photosynthetic apparatus. Effects of iron and copper deficiency on gene expression. To determine whether trace-metal deficiency affects the gene expression, RNA was isolated from cultures grown under different iron and copper conditions (Table 1), and the expression of Lhcx genes was analyzed by qRT-PCR in comparison with the expression of several standard light-harvesting genes (Lhcfs). Steady-state levels of Lhcx1, Lhcx5, and Lhcx6 were all decreased under either iron or copper deficiency, but Lhcx4 was not significantly affected by either treatment (Fig. 1a). Lhcx1 was the most strongly affected, showing a decrease of 1.8-fold under low Cu and 4.5-fold under low Fe. The transcript level of Lhcx5 decreased 2-fold, and that of Lhcx6 decreased 2.5-fold in either the low-Cu or low-Fe treatments. The strongest effect was observed when both Fe and Cu were deficient: 6.7-fold down-regulation of Lhcx1, 2.5-fold of Lhcx5, and 3.7-fold of Lhcx6. This finding suggests that copper deficiency and iron limitation have interactive effects on the expression of the Lhcx genes.


a

b

Fig. 1. Relative expression of Lhcx (a) and Lhcf (b) transcripts when cells were grown under Fe- and Cu-replete, and Fe- and ⁄ or Cu-deplete conditions. Levels of mRNA examined by quantitative real-time PCR were normalized with respect to that of the actin transcript. For each gene, the lowest expression was set to 1. Results from two independent experiments (biological replicates) were shown as mean ± SD. For each gene, error bar represents the standard deviation (SD) of its relative expression in biological duplicate samples.

Expression of the standard light-harvesting gene Lhcf4 was decreased by Cu deficiency alone, but Lhcf5 was little affected (Fig. 1b). Both these genes were down-regulated 2- to 3-fold in the low-iron treatment, and transcript levels were not further decreased when both Fe and Cu were at low levels. By contrast, Lhcf2 did not change significantly under any condition. Effects of iron and copper deficiency on protein expression. To further investigate whether trace-metal deficiency affects the expression of light-harvesting proteins and reaction center proteins, cells grown under the same four culture conditions were analyzed by immunoblotting. We were able to use an

977

antibody specific for the C-terminus of the Lhcx1 protein (Westermann and Rhiel 2005), and an antiserum raised to one of the predominant Lhcf proteins of H. akashiwo (Harnett 1998), which reacts with the major Lhcf proteins of T. pseudonana but not the Lhcx1 protein. In contrast to Lhcx1 transcript, the amount of Lhcx1 protein was up-regulated 2.5-fold under low-Fe conditions and was not significantly affected by low Cu (Fig. 2, a and c). This suggests that the expression of this protein is independently regulated at the transcriptional and translational levels. However, there does appear to be an interactive effect of Fe and Cu since Lhcx1 protein level did not increase as much under low Fe ⁄ low Cu as under low Fe. The level of Lhcf proteins was significantly decreased by both Fe and Cu deficiency, as were the transcript levels of two of the three genes tested in Figure 1. For the D1 protein, used as a proxy for PSII, only small differences were observed under iron or copper deficiency. Since PSI is Fe rich, it was not surprising to find that three PSI core proteins, PsaA ⁄ B, PsaC, and PsaD, were down-regulated under iron-deficiency conditions (Fig. 2b). The almost undetectable level of PsaC under low-iron conditions agrees well with our knowledge of the biochemistry of PsaC, which contains two terminal Fe4S4 clusters (FA and FB) and is the most iron-rich protein in the PSI reaction center. PsaA ⁄ PsaB, the PSI reaction center heterodimer, binds one Fe4S4 cluster (FX); its level is decreased 3-fold under Fe deprivation. PsaD does not contain any iron cofactor but together with PsaC and PsaE forms a stromal ridge on top of PsaA ⁄ B and participates in the docking of the electron acceptor ferredoxin (Amunts and Nelson 2008). The small decrease in PsaD protein levels could be a secondary effect due to the pronounced down-regulation of its neighboring PSI subunits. Chl fluorescence parameters in response to the trace-metal deficiency. The value of Fv ⁄ Fm reflects the maximum quantum yield of PSII and is used as a sensitive indicator of cell photosynthetic performance. Fv ⁄ Fm decreased by 17% under iron deficiency, indicating that iron deprivation had some effect on the PSII reaction center. There was no effect of Cu starvation alone. NPQ reflects the cell’s ability to dissipate excess excitation energy under high-light conditions. As expected, when the actinic light was adjusted to approximate the growth irradiance (150 lmol photons Æ m)2 Æ s)1), only low levels of NPQ were induced in all treatments because the cells were fully acclimated to that light level (Fig. 3b). At higher light intensities (300 and 750 lmol photons Æ m)2 Æ s)1), NPQ increased significantly. The amount of NPQ was approximately the same in control and copper-starved cultures but was lower in the iron-limited cultures (Fig. 3b). This observation shows that the capacity for excess energy dissipation and protection against photoinhibition is decreased

978

SONG-HUA ZHU ET AL.

a

b

c

d

in iron-deficient cultures relative to the copperstarved and control cultures. DISCUSSION

Physiological adaptations: growth rate and cell size. Both Fe and Cu are important micronutrients for marine phytoplankton. Current evidence suggests that there are interactions between these two elements, at least partly because a Cu-binding ferroxidase is part of the Fe-uptake system (Peers et al. 2005, Maldonado et al. 2006, Annett et al. 2008). To study the effects of low Fe or Cu or both on gene expression in the coastal diatom T. pseudonana, we grew the cells with low levels of Fe and ⁄ or Cu. We verified that low Fe has a significant effect on growth rate under our culture conditions, regardless of the Cu level (Table 1). Our results also showed that cell size is decreased under iron limitation. A reduction in cell size with decreasing iron concentrations has been widely observed in coastal and oceanic phytoplankton species including diatoms, dinoflagellates, haptophytes, and green algae (Sunda and Huntsman 1995). The reduced cell size may decrease the cellular iron requirement for growth, through the reduction of iron-containing proteins in essential metabolic pathways and ⁄ or may increase the surface area to volume ratio, which decreases diffusion limitation of iron uptake under iron starvation (Hudson and Morel 1990, Sunda and Huntsman 1995, 1997). Therefore, reduced cell size seems to be an important physiological adaptation for survival in iron-deficient conditions. It appears that Cu deficiency in our media has little effect on cell size in T. pseudonana, which is known to have low Cu requirements compared to oceanic species (Annett et al. 2008). It is important to note that protein content per unit volume is unchanged under any of the nutrient deficiencies, which means

Fig. 2. Protein expression under Fe- and ⁄ or Cu-replete or Feand ⁄ or Cu-deplete conditions assayed by immunoblotting (a, b) and their corresponding densitometric quantifications (c, d) expressed in arbitrary units (A.U.). (a, c) Lhcx1, Lhcf, D1 polypeptides, (b, d) PsaA ⁄ B, PsaC, PsaD polypeptides. Lanes were loaded on an equal protein basis [3 lg Æ lane)1 for (a) or 6 lg Æ lane)1 for (b)]. I and II represent biological duplicates for each treatment.

that an increase in any one protein reflects its increase as a percent of total cellular protein and is not the result of changes in cell biomass. Expression of Lhcx and Lhcf genes under trace-metal deficiency. The expression of three of the Lhcx genes and one of the Lhcf genes was significantly down-regulated under low-Cu conditions. To our knowledge, this is the first report of Cu deficiency affecting the expression of any diatom light-harvesting gene. The same set of genes were even more strongly down-regulated by Fe deficiency, as was an additional gene, Lhcf5. Interactive effects between these two elements are suggested by the observation that the lowest levels of expression are found in the low-Fe ⁄ low-Cu cultures. This is probably due to the presence of a Cu-requiring ferroxidase in the highaffinity Fe transport system (Peers et al. 2005, Maldonado et al. 2006, Annett et al. 2008). The similar expression pattern of both Lhcx and Lhcf genes in cells acclimated to trace-metal deficiency is different from their response to shorter periods (minutes to hours) of high-light stress, where three out of four Lhcx genes were induced but all Lhcf genes were repressed (Zhu and Green 2008, 2010, Zhu 2009). In the present experiments, it was somewhat surprising that the Lhcx genes were repressed under low Fe, because the drastic downregulation of PSI caused by Fe deficiency should result in chronic overreduction of the plastoquinone pool in the same way as high-light exposure. However, there is a lower NPQ level in Fe-deficient cells (Fig. 3b), suggesting they are less able to dissipate excess energy than Fe-sufficient cells. It is interesting to compare our results with those obtained by microarray analysis of T. pseudonana gene expression (Mock et al. 2008). Two Lhcx genes and one Lhcf gene were strongly down-regulated under iron deficiency in their analysis, and similar trends were also observed for cells grown under


a

b

Fig. 3. The maximum quantum yield of PSII (Fv ⁄ Fm) (a) and capacity for light energy dissipation (NPQ) versus irradiance for a fixed illumination of 5 min (b) in Thalassiosira pseudonana. Cells were grown under Fe- and Cu-replete, and Fe- or Cu-starved conditions. Data (±SD) are the average of three independent measurements (biological replicates). NPQ, nonphotochemical quenching.

silicon and nitrogen limitation. However, the microarray analysis showed a higher magnitude decline in these transcripts compared to our data, probably due to the short-term exposure (4 d) of the cells to Fe starvation in their experiments. In our experiments, the cells were grown continuously in exponential phase for a large number of generations and were therefore acclimated to the trace-metaldeficient conditions. Although both Lhcx and Lhcf genes are members of the LHC superfamily, they fall into different clades in the phylogenetic tree (Green 2007), probably reflecting their different functions. It is clear that the Lhcx and Lhcf genes have different expression patterns depending on the type of envi-

979

ronmental stress and the way it is experienced (i.e., short-term exposure vs. long-term growth), which suggests that more than one regulatory pathway is involved in controlling these genes at the transcriptional level. The pennate diatom Phaeodactylum tricornutum can tolerate much more severe Fe limitation than T. pseudonana, more closely resembling the open-ocean species Thalassiosira oceanica, which lives in extremely Fe-limited environments. In P. tricornutum, one Lhcx gene (annotated as Lhcx2) and one typical light-harvesting gene (Lhcf3) were determined to be up-regulated rather than down-regulated under Fe deficiency (Allen et al. 2008). The difference in expression profiles between these two species and between different culture regimes suggests that further work under identical experimental conditions, including light intensity and growth history, is needed to understand the regulation of LHC family members in response to environmental factors. Effect of trace-metal deficiency on photosynthetic proteins. Our results showed that the abundance of PSII reaction center protein D1 is not significantly affected by Fe or Cu stress in T. pseudonana, although the maximum quantum yield of PSII (Fv ⁄ Fm) is somewhat decreased by low Fe. This contrasts with the previous studies, where decreased levels of D1 have been observed under iron starvation in diatoms and green algae (Greene et al. 1992, Geider et al. 1993, Geider and Laroche 1994, Vassiliev et al. 1995). Fe deficiency does decrease NPQ, which should lead to increased photoinhibition (Fig. 3). However, it is possible that our semicontinuous, exponential growth conditions result in less photoinhibitory damage than in other studies, and that the cells are able to repair damaged PSII in a timely fashion, resulting in no detectable decrease in steady-state levels of D1 protein. On the molecular level, PSI appears to be the principle target of iron starvation, probably because the components constituting PSI are enriched in iron (12 Fe per PSI). Our study supports this as several PSI core subunits are strongly depleted under iron-deficient conditions (Fig. 2). Pronounced degradation of PSI proteins under iron stress has previously been shown in green algae, red algae, and cyanobacteria (Moseley et al. 2002, Doan et al. 2003, Kourˇil et al. 2005, Naumann et al. 2005). In addition to the degradation of PSI, there is a remodeling of PSI-associated LHCI in the green alga C. reinhardtii (Moseley et al. 2002, Naumann et al. 2005) and the red alga Rhodella violacea (Doan et al. 2003) under iron deprivation, which could result in decreased efficiency of energy transfer between LHCI and PSI, thereby minimizing the photooxidative stress to the thylakoid membrane. Recent biochemical studies have demonstrated that a few FCPs are tightly bound to PSI and can be isolated as a PSI-FCP supercomplex in the pennate diatom P. tricornutum (Veith and Bu¨chel 2007) and

980

SONG-HUA ZHU ET AL.

in the centric diatom Chaetoceros gracilis (Ikeda et al. 2008). In C. meneghiniana, one of these polypeptides was identified as the Lhcr-type using a specific antibody and mass spectroscopy (Veith et al. 2009). If some of the ‘‘Lhcf’’ polypeptides detected by our anti-HaFCP antibody (Fig. 2) were associated with PSI, this would be consistent with their down-regulation under iron deficiency and could contribute to the remodeling of the photosynthetic apparatus under iron deprivation. On the other hand, the accumulation of the Lhcx1 polypeptide was more than doubled in response to iron deficiency in T. pseudonana. This protein is the homologue of the Fcp6 protein of C. meneghiniana, which appears to be associated with PSII (Veith et al. 2009). Lhcx1 is accumulated in a stable fashion under high-light stress conditions (i.e., it does not appear to turn over rapidly) (Zhu 2009, Zhu and Green 2010). It may therefore not require a high level of transcription under chronic iron deficiency. If it is associated with PSII, it could be involved in stabilizing PSII under conditions of decreased thermal dissipation (Fig. 3b). In summary, our results show that under both lowCu and low-Fe growth conditions, the transcription of two kinds of light-harvesting gene (Lhcx and Lhcf) is diminished. Fe deprivation dramatically decreases the level of PSI core proteins PsaA ⁄ B and PsaC but has the opposite effect on the level of Lhcx1 protein, although this effect is modulated under Cu deficiency. Taken together with our work on high-light stress (Zhu and Green 2010), it is clear that at least some of the Lhcx genes are stress-response genes, but their exact roles in responding to the different types of environmental stress remain to be determined. This research was supported by the Natural Sciences and Engineering Council of Canada. Allen, A. E., LaRoche, J., Maheswari, U., Lommer, M., Schauer, N., Lopez, P. J., Finazzi, G., Fernie, A. R. & Bowler, C. 2008. Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc. Natl. Acad. Sci. U. S. A. 105: 10438–43. Amunts, A. & Nelson, N. 2008. Functional organization of a plant photosystem I: evolution of a highly efficient photochemical machine. Plant Physiol. Biochem. 46:228–37. Annett, A. L., Lapi, S., Ruth, T. J. & Maldonado, M. T. 2008. The effects of Cu and Fe availability on the growth and Cu:C ratios of marine diatoms. Limnol. Oceanogr. 53:2451–61. Armbrust, E. V., Berges, J. A., Bowler, C., Green, B. R., Martinez, D., Putnam, N. H., Zhou, S. G., et al. 2004. The genome of the diatom Thalassiosira pseudonana: ecology, evolution, and metabolism. Science 306:79–86. Bilger, W. & Bjorkman, O. 1990. Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbency changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth. Res. 25:173–85. Boyd, P. W., Watson, A. J., Law, C. S., Abraham, E. R., Trull, T., Murdoch, R., Bakker, D. C. E., et al. 2000. A mesoscale phytoplankton bloom in the polar Southern Ocean stimulated by iron fertilization. Nature 407:695–702. Brand, L. E., Guillard, R. R. L. & Murphy, L. S. 1981. A method for the rapid and precise determination of acclimated phytoplankton reproduction rates. J. Plankton Res. 3:193–202.

Bruland, K. W., Donat, J. R. & Hutchins, D. A. 1991. Interactive influences of bioactive trace-metals on biological production in oceanic waters. Limnol. Oceanogr. 36:1555–77. Chadd, H. E., Newman, J., Mann, N. H. & Carr, N. G. 1996. Identification of iron superoxide dismutase and a copper ⁄ zinc superoxide dismutase enzyme activity within the marine cyanobacterium Synechococcus sp. WH 7803. FEMS Microbiol. Lett. 138:161–5. Coale, K. H., Johnson, K. S., Fitzwater, S. E., Gordon, R. M., Tanner, S., Chavez, F. P., Ferioli, L., et al. 1996. A massive phytoplankton bloom induced by an ecosystem-scale iron fertilization experiment in the equatorial Pacific Ocean. Nature 383:495–501. Debaar, H. J. W., Dejong, J. T. M., Bakker, D. C. E., Loscher, B. M., Veth, C., Bathmann, U. & Smetacek, V. 1995. Importance of iron for plankton blooms and carbon-dioxide drawdown in the Southern Ocean. Nature 373:412–5. Doan, J. M., Schoefs, B., Ruban, A. V. & Etienne, A. L. 2003. Changes in the LHCI aggregation state during iron repletion in the unicellular red alga Rhodella violacea. FEBS Lett. 533: 59–62. Gagne, G. & Guertin, M. 1992. The early genetic response to light in the green unicellular alga Chlamydomonas eugametos grown under light dark cycles involves genes that represent direct responses to light and photosynthesis. Plant Mol. Biol. 18:429– 45. Geider, R. J. & Laroche, J. 1994. The role of iron in phytoplankton photosynthesis, and the potential for iron-limitation of primary productivity in the sea. Photosynth. Res. 39:275–301. Geider, R. J., Laroche, J., Greene, R. M. & Olaizola, M. 1993. Response of the photosynthetic apparatus of Phaeodactylum tricornutum (Bacillariophyceae) to nitrate, phosphate, or iron starvation. J. Phycol. 29:755–66. Green, B. R. 2003. The evolution of light-harvesting antennas. In Green, B. R. & Parson, W. W. [Eds.] Light-harvesting Antennas in Photosynthesis. Kluwer Academic, Dordrecht, the Netherlands, pp. 129–68. Green, B. R. 2007. Evolution of light-harvesting antennas in an oxygen world. In Falkowski, P. G. & Knoll, A. H. [Eds] Evolution of Primary Producers in the Sea. Elsevier, Burlington, Massachusetts, pp. 37–53. Greene, R. M., Geider, R. J., Kolber, Z. & Falkowski, P. G. 1992. Iron-induced changes in light harvesting and photochemical energy-conversion processes in eukaryotic marine algae. Plant Physiol. 100:565–75. Harnett, J. P. 1998. Isolation and characterization of photosystem I and the fucoxanthin-chlorophyll a ⁄ c proteins of the chromophyte alga Heterosigma carterae. MSc dissertation, University of British Columbia, Vancouver, 111 pp. Hudson, R. J. M. & Morel, F. M. M. 1990. Iron transport in marinephytoplankton – kinetics of cellular and medium coordination reactions. Limnol. Oceanogr. 35:1002–20. Ikeda, Y., Komura, M., Watanabe, M., Minami, C., Koike, H., Itoh, S., Kashino, Y. & Satoh, K. 2008. Photosystem I complexes associated with fucoxanthin-chlorophyll-binding proteins from a marine centric diatom, Chaetoceros gracilis. Biochem. Biophys. Acta Bioenergetics 1777:351–61. Im, C. S., Zhang, Z. D., Shrager, J., Chang, C. W. & Grossman, A. R. 2003. Analysis of light and CO2 regulation in Chlamydomonas reinhardtii using genome-wide approaches. Photosynth. Res. 75:111–25. Johnson, K. S., Gordon, R. M. & Coale, K. H. 1997. What controls dissolved iron concentrations in the world ocean? Mar. Chem. 57:137–61. Kourˇil, R., Arteni, A. A., Lax, J., Yeremenko, N., D’Haene, S., Ro¨gner, M., Matthijs, H. C. P., Dekker, J. P. & Boekema, E. J. 2005. Structure and functional role of supercomplexes of IsiA and photosystem I in cyanobacterial photosynthesis. FEBS Lett. 579:3253–7. Koziol, A. G., Borza, T., Ishida, K. I., Keeling, P., Lee, R. W. & Durnford, D. G. 2007. Tracing the evolution of the light-harvesting antennae in chlorophyll a ⁄ b-containing organisms. Plant Physiol. 143:1802–16.

F e - C u D E F I C I E N C Y A N D L h c E X PR E S S I O N La Fontaine, S., Quinn, J. M., Nakamoto, S. S., Page, M. D., Go¨hre, V., Moseley, J. L., Kropat, J. & Merchant, S. 2002. Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote Chlamydomonas reinhardtii. Eukaryot. Cell 1:736–57. Maheswari, U., Montsant, A., Goll, J., Krishnasamy, S., Rajyashri, K. R., Patell, V. M. & Bowler, C. 2005. The diatom EST database. Nucleic Acids Res. 33:D344–7. Maldonado, M. T., Allen, A. E., Chong, J. S., Lin, K., Leus, D., Karpenko, N. & Harris, S. L. 2006. Copper-dependent iron transport in coastal and oceanic diatoms. Limnol. Oceanogr. 51:1729–43. Martin, J. H., Coale, K. H., Johnson, K. S., Fitzwater, S. E., Gordon, R. M., Tanner, S. J., Hunter, C. N., et al. 1994. Testing the iron hypothesis in ecosystems of the equatorial Pacific Ocean. Nature 371:123–9. Martin, J. H., Gordon, R. M. & Fitzwater, S. E. 1990. Iron in Antarctic waters. Nature 345:156–8. Merchant, S. S., Allen, M. D., Kropat, J., Moseley, J. L., Long, J. C., Tottey, S. & Terauchi, A. M. 2006. Between a rock and a hard place: trace element nutrition in Chlamydomonas. Biochem. Biophys. Acta Mol. Cell Res. 1763:578–94. Mock, T., Samanta, M. P., Iverson, V., Berthiaume, C., Robison, M., Holtermann, K., Durkin, C., et al. 2008. Whole-genome expression profiling of the marine diatom Thalassiosira pseudonana identifies genes involved in silicon bioprocesses. Proc. Natl. Acad. Sci. U. S. A. 105:1579–84. Moseley, J. L., Allinger, T., Herzog, S., Hoerth, P., Wehinger, E., Merchant, S. & Hippler, M. 2002. Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus. EMBO (Eur. Mol. Biol. Organ.) J. 21:6709–20. Moseley, J. L., Chang, C. W. & Grossman, A. R. 2006. Genomebased approaches to understanding phosphorus deprivation responses and PSR1 control in Chlamydomonas reinhardtii. Eukaryot. Cell 5:26–44. Naumann, B., Busch, A., Allmer, J., Ostendorf, E., Zeller, M., Kirchhoff, H. & Hippler, M. 2007. Comparative quantitative proteomics to investigate the remodeling of bioenergetic pathways under iron deficiency in Chlamydomonas reinhardtii. Proteomics 7:3964–79. Naumann, B., Stauber, E. J., Busch, A., Sommer, F. & Hippler, M. 2005. N-terminal processing of Lhca3 is a key step in remodeling of the photosystem I light-harvesting complex under iron deficiency in Chlamydomonas reinhardtii. J. Biol. Chem. 280:20431–41. Peers, G., Quesnel, S. A. & Price, N. M. 2005. Copper requirements for iron acquisition and growth of coastal and oceanic diatoms. Limnol. Oceanogr. 50:1149–58. Price, N. M., Harrison, G. I., Hering, J. G., Hudson, R. J., Nirel, P. M. V., Palenik, B. & Morel, F. M. M. 1988 ⁄ 1989. Preparation and chemistry of the artificial algal culture medium Aquil. Biol. Oceanogr. 6:443–62. Raven, J. A. 1990. Predictions of Mn and Fe use efficiencies of phototrophic growth as a function of light availability for growth and of C assimilation pathway. New Phytol. 116:1–18. Raven, J. A., Evans, M. C. W. & Korb, R. E. 1999. The role of trace metals in photosynthetic electron transport in O2-evolving organisms. Photosynth. Res. 60:111–49. Sandmann, G., Reck, H., Kessler, E. & Boger, P. 1983. Distribution of plastocyanin and soluble plastidic cytochrome c in various classes of algae. Arch. Microbiol. 134:23–7.

981

Savard, F., Richard, C. & Guertin, M. 1996. The Chlamydomonas reinhardtii LI818 gene represents a distant relative of the cab I ⁄ II genes that is regulated during the cell cycle and in response to illumination. Plant Mol. Biol. 32:461–73. Schreiber, U., Endo, T., Mi, H. L. & Asada, K. 1995a. Quenching analysis of chlorophyll fluorescence by the saturation pulse method-particular aspects relating to the study of eukaryotic algae and cyanobacteria. Plant Cell Physiol. 36:873–82. Schreiber, U., Hormann, H., Neubauer, C. & Klughammer, C. 1995b. Assessment of photosystem II photochemical quantum yield by chlorophyll fluorescence quenching analysis. Aust. J. Plant. Physiol. 22:209–20. Stryer, L. 1988. Biochemistry. W. H. Freeman, New York, 1089 pp. Sunda, W. G. & Huntsman, S. A. 1995. Iron uptake and growth limitation in oceanic and coastal phytoplankton. Mar. Chem. 50:189–206. Sunda, W. G. & Huntsman, S. A. 1997. Interrelated influence of iron, light and cell size on marine phytoplankton growth. Nature 390:389–92. Tsuda, A., Takeda, S., Saito, H., Nishioka, J., Nojiri, Y., Kudo, I., Kiyosawa, H., et al. 2003. A mesoscale iron enrichment in the western subarctic Pacific induces a large centric diatom bloom. Science 300:958–61. Vassiliev, I. R., Kolber, Z., Wyman, K. D., Mauzerall, D., Shukla, V. K. & Falkowski, P. G. 1995. Effects of iron limitation on photosystem II composition and light utilization in Dunaliella tertiolecta. Plant Physiol. 109:963–72. Veith, T., Brauns, J., Weisheit, W., Mittag, M. & Bu¨chel, C. 2009. Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana. Biochem. Biophys. Acta Bioenergetics 1787:905–12. Veith, T. & Bu¨chel, C. 2007. The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes. Biochem. Biophys. Acta Bioenergetics 1767:1428–35. Westermann, M. & Rhiel, E. 2005. Localisation of fucoxanthin chlorophyll a ⁄ c-binding polypeptides of the centric diatom Cyclotella cryptica by immuno-electron microscopy. Protoplasma 225:217–23. Yamano, T., Miura, K. & Fukuzawa, H. 2008. Expression analysis of genes associated with the induction of the carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol. 147:340–54. Zhang, Z. D., Shrager, J., Jain, M., Chang, C. W., Vallon, O. & Grossman, A. R. 2004. Insights into the survival of Chlamydomonas reinhardtii during sulfur starvation based on microarray analysis of gene expression. Eukaryot. Cell 3:1331–48. Zhu, S. H. 2009. Characterization of LI818-like genes under various stress conditions in the marine diatom Thalassiosira pseudonana. PhD dissertation, University of British Columbia, Vancouver, 190 pp. Zhu, S. H. & Green, B. R. 2008. Light-harvesting and photoprotection in diatoms: identification and expression of LI818-like proteins. In Allen, J. F., Gantt, E., Golbeck, J. H. & Osmond, B. [Eds.] Photosynthesis. Energy from the Sun. Springer, Heidelberg, Germany, pp. 261–4. Zhu, S. H. & Green, B. R. 2010. Photoprotection in the diatom Thalassiosira pseudonana: role of LI818-like proteins in response to high light stress. Biochem. Biophys. Acta Bioenergetics 1797:1449–57.